The PEG-responding desiccome of the alder microsymbiont Frankia alni

International audience; Actinorhizal plants are ecologically and economically important. Symbiosis with nitrogen-fixing bacteria allows these woody dicotyledonous plants to colonise soils under nitrogen deficiency, water-stress or other extreme conditions. However, proteins involved in xerotolerance of symbiotic microorganisms have yet to be identified. Here we characterise the polyethylene glycol (PEG)-responding desiccome from the most geographically widespread Gram-positive nitrogen-fixing plant symbiont, Frankia alni, by next-generation proteomics, taking advantage of a Q-Exactive HF tandem mass spectrometer equipped with an ultra-high-field Orbitrap analyser. A total of 2,052 proteins were detected and quantified. Under osmotic stress, PEG-grown F. alni cells increased the abundance of envelope-associated proteins like ABC transporters, mechano-sensitive ion channels and Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-associated (cas) components. Conjointly, dispensable pathways, like nitrogen fixation, aerobic respiration and homologous recombination, were markedly down-regulated. Molecular modelling and docking simulations suggested that the PEG is acting on Frankia partly by filling the inner part of an up-regulated osmotic-stress large conductance mechanosensitive channel. Actinobacteria belonging to the genus Frankia do establish nitrogen-fixing nodular symbiosis with the roots of 23 angiosperm genera that are collectively called " actinorhizals " 1. These plants form root nodules in which Frankia fixes nitrogen, thus permitting them to thrive in pioneer soils poor in nitrogen and organic matter, such as glacial moraines, lava fields, forest burnouts or anthropogenic sites such as mine spoils or hydrodam dykes 2. Frankia establishes a symbiotic association with the roots of several dicotyledonous plants. The different Frankia lineages form a coherent cluster at the root of the aerobic actinobacteria phylum 3 , and F. alni in particular establishes symbiosis with alder (Alnus) and bayberry (Morella) species 4. The interaction has evolved over several million years with a sophisticated dialogue that does not imply acy-lated N-acetyl-glucosamine oligomeric Nod factors 5. The Frankia determinants of symbiosis are still poorly known, which is for the most part due to the lack of a genetic transformation system. Transcriptomics has shown genes coding for nitrogenase (nif), hydrogenase uptake (hup), hopanoids (shc, hpn), iron-sulfur (suf) clusters as among the most up-regulated 6. Proteomics has also been used to analyze the symbiosis 7 to identify up-regulated peptides and it has shown the presence of Nif, Hup, Suf, Hop proteins as expected but also several transporters, regulators and various proteins involved in stress responses.